The long-term objectives of this research plan are to combine approaches of neuroanatomy, cellular biology and mouse behavior tests to probe for mechanisms regulating optic nerve regeneration and restoring vision after injury or diseases. Optic nerve damage is a major feature of several blinding diseases, such as glaucoma, optic neuropathy, and trauma. Finding ways to rescue retinal ganglion cells from degeneration and to promote optic nerve regrowth are crucial to the development of efficacious treatment for these conditions. Previously, using the established model of optic nerve axotomy and mouse genetic technology, we identified two barriers to optic nerve regeneration the loss of Bcl-2 expression and maturation of astrocytes and their acquisition of the ability to form glial scars. By eliminating these two barriers demonstrated for the first time successful optic nerve re-elongation into the brain targets, up to postnatal day 14 (P14) in mice. Interestingly, regenerating axons in the postnatal mice entered the wrong (ipsilateral) side of the brain and innervated the visual targets in there. However, the regeneration fails consistently if the injury is incurred after P14, suggestion development of additional barriers to nerve regrowth at this age. The key remaining questions are: (1) what is the additional barrier to optic nerve regeneration that is developed after P14 and whether successful regeneration of the severed optic nerve can be achieved in the adult, and (2) whether regenerated axons are capable of establishing a topographic map and functional synapses with target neurons and in turn restoring vision. Recent studies in my laboratory implicate that appearance of the third and likely the last barrier to optic nerve regeneration is related to the development of CNS myelin. We hypothesize that the three barriers to optic nerve regeneration include the loss of Bcl-2 expression by retinal ganglion cells, development of glial scars, and appearance of CNS myelin;simultaneous elimination of the three barriers will allow optic nerve regeneration and restoration of vision in the adult. The three specific aims of this research plan includes: (1) To determine if simultaneous eliminating the three barriers to retinal ganglion axonal regrowth will enable optic nerve regeneration in the adult. (2) To determine a role for EphB1 in directing the ipsilateralization projection of retinal ganglion cell axons during regeneration, using the model of EphB1 knockout mice. (3) To investigate whether regenerated RGC axons are capable of establishing topographically ordered functional connections that can lead to vision restoration, using neuroanatomy, visual evoked potential, and animal behavioral tests. The results of this study may lead to a novel strategy for using neural transplantation to treat blindness and other CNS diseases. Project Narrative: Failure of optic nerve injury to be repaired is a major feature of several blinding diseases, including glaucoma, optic neuritis, and multiple sclerosis, as well as trauma. Our present research plan is proposed to identify and alleviate all barriers to optic nerve regeneration. The results of these proposed studies will open the door for future development of treatment strategies for optic nerve damage after disease or injury.